Immunological test kit with immunologically invisible carrier

ABSTRACT

Immunologically invisible carrier molecules connect a plurality of copies of an immunologically active molecule in an immunologic assay.

CROSS REFERENCES

[0001] This application claims priority from Stanley STEIN et al.,“Highly Sensitive and Specific IgM-Capture . . . ,” provisional patentfiling serial No. 60/242,819, filed Oct. 24, 2000 and Bo QUI et al.,“Multiple Epitopes Connected By A Carrier,” Ser. No. ______, filedOctober 2001. The contents of these, together with Bo QUI, “Studies onPolymers” (unpublished) and Bo QIU et al., “Selection of ContinuousEpitope Sequences,” 55 Biopolymers 319 (2001) are incorporated here byreference.

GOVERNMENT RIGHTS

[0002] There are no Federal rights in this invention.

BACKGROUND

[0003] Current technology enables correct diagnosis of certaininfectious diseases only after the disease has progressed to a certainmaturity. By that time, however, treatment is more difficult. We havefound a way to make disease diagnosis, even at an early stage, much moresensitive.

SUMMARY

[0004] Our invention entails presenting an immunologically reactivesubstance (e.g., epitope polypeptide) in multiple copies conjugated toan immunologically invisible carrier.

[0005] This basic conjugate has a variety of versions or embodiments.For example, while we do not prefer it, the epitope can be substitutedor supplemented with any immunologically reactive substance such as anepitope, antigen (e.g., a polypeptide or nucleic acid) or antibody.Similarly, we prefer the carrier also connect a reporter moiety to makedetection of the conjugate simpler.

[0006] The conjugate so made may then be used in a variety of ways. Forexample, we have shown it effective as part of an immunological assay.Alternatively, the conjugate may be used as a vaccine. Alternatively,the conjugate may be used as an in vivo therapeutic.

[0007] Thus, our basic idea can be used to make, for example, animmunological test kit. The term “immunological test kit” means a testkit which uses immune (e.g., antibody-epitope or antibody-antigen)interaction to test for the presence or absence of an anlayte.Currently-known examples include ELISA, capillary immuno-chromatographyand column immuno-chromatography. In making an immunological test kit,it may be desirable to conjugate a reporter moiety on theimmunologically invisible carrier (e.g., polyethylene glycol). Asanother example, our basic idea can be used to conjugate severalimmunologically reactive substances (either several copies of the samesubstance, or copies of each of several different substances) togetherusing an immunologically invisible carrier, which conjugate can be thenused in an immunological test kit.

[0008] The immunologically reactive substance(s) can be one or more ofthe Borellia burgdorferii epitope polypeptides we discovered:VQEGVQQEGAQQP-(beta-A)(beta-, 4)C; EIAAKAIGKKIHQNNG-(beta-A)(beta-A)C;ISTLIKQKLDGLKNE-(beta-A)(beta-A)C; PWAESPKKPE-(beta-A)(beta-A)C;DKKAINLDKAQQKLD-(beta-A)(beta-A)C; ITKGKSQKSLGD-(beta-A)(beta-A)C; andGMTFRAQEGAFLTG-(beta-A)(beta-A)C. Alternatively, one could use asantigen the nucleic acid coding for one or more of these epitopes. Usingsuch an epitope enables one to make an apparatus for isolatinganti-Borellia burgdorferi antibody (i.e., a Lyme disease test kit), avaccine, or a therapeutic. Similarly, the nucleic acid sequences codingfor these polypeptides may be useful as antigen, or to make largequantity of polypeptide.

[0009] Our basic idea can be made using, as an immunologically invisiblecarrier, a polyethylene glycol copolymer that we invented. It has thestructure:

[0010] We prefer using such a polyethylene glycol copolymer with thestructure:

[0011] These are some of the many variations on our basic theme. Inwhatever variation, however, our invention ultimately requirespresenting one or more immunologically reactive substances (e.g.,epitope polypeptides) connected by an immunologically invisible carrier.We now discuss each of the components of our invention in turn.

[0012] Immunologically Reactive Substance

[0013] Antibodies generally cannot bind to the whole antigen molecule.Rather, a specific antibody binds specifically to one individual epitopeon that antigen. The term “immunologically reactive substance” means anepitope, and antigen or an antibody. To increase the specificity of ourassay, we prefer to use not entire antigens, but one or more definedepitopes.

[0014] The success of a specific and sensitive immunoassay largelydepends on the strength of antigen-antibody binding and the stability ofthe complex formed between the antigen and the antibody. The strength ofantigen-antibody binding is measured by affinity, an intrinsic propertyof an antigen for a given antibody. To select an epitope peptide is toidentify a peptide sequence with high affinity that can bind stronglywith specific antibodies.

[0015] The stability of complex between antigen and antibody is measuredby avidity, which is determined by three factors, the intrinsic affinityof the antibody for the antigen, the valence of the antibody andantigen, and the geometric arrangement of the interacting components.Thus, our invention works best when affinity, avidity and specificity(e.g., cross-reactivity) are used to first select an appropriateepitope(s). After the specific epitopes are selected, they can be madeas desired (e.g., purified from natural protein or synthesized).

[0016] The sensitivity of an immunoassay relies on providing enough ofeach epitope and on having the right orientation and conformation of theepitope. Thus, we prefer the epitope peptides be modified as necessaryto assume the right orientation and conformation to obtain a strongantigen-antibody binding.

[0017] Whole antigen or antibody may be used instead of epitope, tomount to the carrier molecule. If mounting antibody on the carrier, theantibody-carrier complex can be used to trap antigen or epitope analytein the teat solution.

[0018] Multiple Copies

[0019] Epitopes are specific, but have a key shortcoming. The affinityof epitope peptides to anti-protein antibodies can be 100 to 1,000 timesweaker than that of the whole antigen (whole protein). Thus, theaffinity between a single epitope and the serum antibody might not bestrong enough to endure the vigorous washing steps in an immunoassay.

[0020] To address this problem, we use multiple copies of each epitope,connected together with a “carrier.” Connecting multiple copies ofepitope peptides enable the epitopes to form multivalent interactionsbetween two or more Fab fragments of the antibody. This creates asynergistically greater binding strength. More specifically, bindingstrength increases, perhaps exponentially, with the number of additionalcopies of epitope connected to the carrier.

[0021] For example, an epitope alone may have an antibody affinity 100times weaker than the native antigen. The same epitope, however, ifprovided in pairs (i.e., two copies of the epitope connected together),might have affinity only 10 times weaker than the native antigen.Further, the same epitope provided in trios (i.e., three copies of theepitope connected together) might have native-strength affinity. Webelieve this effect especially true where the target antibody is IgM,itself a pentamer.

[0022] Immunologically Invisible Carrier

[0023] We call the material that connects the various copies of theepitope a “carrier” molecule. Any molecule that can bind more than onecopy of an epitope can function as a “carrier.” Examples include keyholelimpet hemacyanin, albumins such as serum albumin (e.g., bovine serumalbumin, mouse serum albumin, rabbit serum albumin) and ovalbumin, andpolyethylene glycol derivatives. These materials can each bind multiplecopies of an epitope.

[0024] Of these carriers, however, most are unsuitable because they areimmunologically “visible,” that is to say, they react in animmunological test (even without epitope present) to create astatistically significant increase in (sometimes random) backgroundreactivity. Albumin and limpet hemacyanin tend to stick to ELISA plates.Thus, when using these proteins as carriers, the carrier itself adheresto the ELISA plate in quantity sufficient to cause an elevatedbackground. This problem is particularly significant in developingdiagnostic assays for disease where the serum antibody level isrelatively low and the signals thus barely detectable. The elevatedbackground compromises the signals, ruining the assay sensitivity andspecificity.

[0025] Our invention is thus limited to “immunologically invisible”carriers. Excluded from the term “immunologically invisible” are fulllength albumins and keyhole limpet hemacyanin, because these are notimmunologically “invisible.”

[0026] Biocompatible Polymers

[0027] Immunologically invisible carriers are carriers which do notgenerate statistically significant background immunological reactivity.Immunologically invisible carriers include, for example, biocompatiblepolymers.

[0028] Such polymers are known in the art. General reviews of suchcompounds include Langer, R., “Biomaterials in Drug Delivery,” 33ACC.CHEM.RES. 94 (2000); and Langer, R., “Tissue Engineering,” 1MOL.THER. 12 (2000). One example of such an immunologically invisiblecompound is a N-vinylpyrrolidone-methyl methacrylate co-polymer, perhapswith added polyamide-6. Buron, F. et al., Biocompatable OsteoconductivePolymer, 16 CLIN.MATER. 217 (1994). Another example ispoly(DL-lactide-co-glycolide) capsules. Isobe, M. et al., BoneMorphogenic Protein Encapsulated with a Biodegradable and BiocompatiblePolymer, 32 J.BIOMED.MATER.RES. 433 (1996). Another example is a 70:30ratio mixture of methylmethacrylate:2-hydroxyethyl methacrylate. Bar, F.W. et al., New Biocompatable Polymer Surface Coating, 52J.BIOMED.MATER.RES. 193 (2000). Another example is2-methacryloyloxyethyl phosphorylcholine, perhaps with polyurethane.Iwasaki, Y. et al., Semi-Interpenetrating Polymer Networks . . . , 52J.BIOMED.MATER.RES. 701 (2000). Polyvinyl pyrolidone may also be used,as may polyethylene glycol and its derivatives. Other biocompatiblepolymenrs are known in the art. E.g., Haisch, A. et al., TissueEngineering of Human Cartilage Tissue, 44 HNO 624 (1996); Ershov, I. A.et al., Polymer Biocompatible X-Ray Contract Hydrogel, 2 MED.TEKH. 37(1994); Polous, I. M. et al., Use of A Biocompatible AntimicrobialPolymer Film, 134 VESTN.KHIR.IM.II GREK. 55 (1985).

[0029] In addition to such synthetic polymers, immunologically invisiblebiological materials may be used. An example is calcium alginate, suchas purified high guluronic acid alginates. Becker, T. A. et al., CalciumAlginate Gel, 54 J.BIOMED.MATER.RES. 76 (2001). Genetically engineeredprotein polymers also may be acceptable. Buchko, C. J. et al., SurfaceCharacterization of Porous, Biocompatible Protein Polymer Thin Films, 22BIOMATERIALS 1289 (2001); cf. Raudino, A. et al., Binding of LipidVescicles . . . , 231 J.COLLOID.INTERFACE SCI. 66 (2000).

[0030] Such compounds may lack functional groups useful for attachingthe desired immunologically reactive substance to the carrier. Thus, itmay be desirable to use not the pure polymer, but a co-polymer havingappended functional groups. The functional groups may then be filledwith the desired immunologically reactive substance.

[0031] As immunologically invisible carrier, we prefer polyethyleneglycol and its derivatives. We thus now discuss it in some detail.

[0032] Polyethylene Glycol

[0033] Polyethylene glycol (often simply called “PEG”) is a watersoluble, non-immunogenic, biocompatible material. When used as acarrier, the useful properties of polyethylene glycol with respect tothe appended moiety include improved solubility, increased circulationlifetime in bloodstream, resistance to proteases and nucleases, etc. Thelarge molecular weight of polyethylene glycol makes it very easy toseparate the final conjugates from excess epitope peptide and othersmall-size impurities. Polyethylene glycol does not aggregate, degradeor denature. Polyethylene glycol conjugates are thus stable andconvenient for use in diagnostic assays.

[0034] While the polyether backbone of polyethylene glycol is chemicallyinert, the primary hydroxyl groups on both ends are reactive and can beutilized directly to attach immunologically reactive substances. Thesehydroxyl groups have been transformed into more reactive functionalgroups for conjugation purposes. Such polyethylene glycol derivativespossess only two functional groups on the ends. This limits the numberof conjugations to just two. We thus prefer a polyethylene glycolderived polymer system with multiple functional groups for epitopepeptide attachment.

[0035] We made a new polyethylene glycol with multiple functional groupsand a favorable geometric arrangement to achieve strong and stableantigen-antibody blinding for the selected epitope peptides. We usedα,ω-diamino-polyethylene glycol to copolymerize with aminogroup-protected aspartic acid to obtain a new polyethyleneglycol-aspartic acid copolymer. Multiple attachment sites becomeavailable for conjugation through the pendant amino groups of theaspartic acid residue upon removal of the protection (FIG. 1).

[0036] To allow the attached epitope peptides to assume a favorablegeometric arrangement for antibody binding, we used a long armcross-linker for attaching the epitope peptides to the amino groups, sothat the attached epitope peptides can be positioned far enough from thepolymer backbone to avoid steric hindrance. We used a heterobifunctionalpolyethylene glycol-based cross-linker, NHS-polyethylene glycol-VS, asthe cross-linker for epitope peptide conjugation.

[0037] The conjugation of epitope peptides may use thiol-specificchemistry under mild conditions. The easiest strategy for peptideconjugation is to add an extra amino acid on either the ammo or carboxylterminus of the peptide to allow one-site coupling to the carrier. Inour study design, a cysteine residue, followed by two β-alanineresidues, was incorporated at the C-terminus of each epitope peptideduring solid phase peptide synthesis. Putting two more β-alanineresidues between the conjugation anchor, cysteine, and the epitopepeptide is used as a precaution to generate further flexibility of thelinear peptides, and therefore help them to adopt the optimalconformations for stronger antibody binding. The N-terminus of thepeptides needs to be capped in order to remove charges associated withfree amino groups and thereby mimicking the real environment in theprotein.

[0038] To conjugate epitope peptides to the polymer backbone, a two stepapproach can be used. A heterobifunctional cross-linker,NHS-polyethylene glycol-VS can first react with the reserved aminogroups in the reporter-labeled polymer carrier through, the NHS groups.After purification to remove excess cross-linker, cysteine-containingepitope peptides can then react readily with vinylsulfone groups (VS) tocomplete the conjugation. The final polyethylene glycol-peptideconjugates containing multiple copies of epitope peptides and severalcopies of reporter molecules are now ready for immunoassays (FIG. 2).

[0039] Reporter

[0040] The carrier-epitope conjugates may be labeled by, for example,washing with labeled anti-epitope antibody. Alternatively, a label or“reporter” moiety may be conveniently included in the carrier-epitopeconjugates; this allows for a one-step (rather than a two-step)detection process. The construction of such carrier-epitope conjugatesinvolves two aspects: the conjugation of reporter molecules, and theconjugation of epitope peptides.

[0041] A commonly used reporter molecule in immunoassay is biotin. Itscorresponding N-hydroxysuccinimide ester (NHS) with extended spacer ischosen for our carrier-peptide conjugate preparation. We did thisbecause the NHS group can react readily with the pendant amino groups ofthe polyethylene glycol-aspartic acid copolymer under mild conditions.The extended spacer arm can help lower steric hindrance and thusfacilitate assay detection. Since biotin detection system is extremelysensitive, a few label molecules should suffice to give satisfactorysignals. Therefore, only a small portion of attachment sites in thecarrier is needed to attach reporter molecules so that a large portionof the attachment sites can be reserved for the epitope peptides togenerate polyvalent antigen with improved antibody binding and toimprove the sensitivity of the immunoassay.

[0042] Alternatively, the reporter molecule can be put on the N-terminusof the epitope peptides during the solid phase peptide synthesis. Thereporter molecules can thus serve as the capping groups of the peptidesand as the reporter groups of the conjugates simultaneously. By puttingthe reporter groups both on the polymer backbone and on the epitopepeptides, the assay signal can be further enhanced (FIG. 3). Care mustbe taken to not block the epitope from contacting and binding to theantibody. Multiple copies of the reporter groups attached to the carrieramplify the assay signal. Other reporters or labels (e.g., colloidalmetal, carbon black, latex beads) are known in the art and mayalternatively be used.

[0043] Uses

[0044] Once made, our carrier-epitope conjugates can be used for avariety of things. For example, our conjugates can be used inimmuno-chromatography, the specific kind of chromatography selecteddepending on one's goals. Column chromatography, for example, can bedone with our conjugates used to isolate and purify a desired antibodyin quantity. Alternatively, capillary chromatography can be done withour conjugates, to detect low levels of antibody in a sample. Similarly,ELISA can be done with our conjugates, to detect low levels of antibodyin a clinical sample. We actually used our conjugates to make such animmunodiagnostic kit, so we will now discuss how to make such a kit insome detail.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0045] Our preferred embodiment of our invention entails four parts: 1)the selection of specific epitopes by epitope mapping; 2) the design andsynthesis of a carrier molecule with multiple attachment sites; 3) thepreparation of multivalent carrier-peptide conjugates with one or morereporter groups; and 4) the use of the prepared carrier-peptide-reporterconjugates in an immunological assay. Here is how you can use of ourpreferred embodiment to make an indirect IgM-capture ELISA effective forthe diagnosis of Lyme disease at its earliest stage.

[0046] Epitope Mapping by SPOTS

[0047] All concurrent peptide sequences were generated using computersoftware provided by the manufacturer (Genesys) with the SPOTS kit. Byproviding a protein sequence, desired length of each peptide and offsetof amino acids for each peptide, the program can edit peptide sequencesto be assembled on SPOTS membrane and provide the amino acid additionschedule for each synthesis cycle.

[0048] To start the peptide synthesis on the membrane, pre-weighedFmoc-amino acid active esters were dissolved in DMF and pipetted toappropriate spots on the membrane based on the generated synthesisschedule. Double coupling was done for each cycle to ensure thecompletion of the reaction. All the Fmoc-amino acid active esters,except Arginine, are relatively stable and can be dissolved in DMF foruse of several cycles in the same working day, as long as they arestored at −20° C. between each addition. Due to its intrinsicinstability, the Fmoc-Arginine active ester must be dissolved justbefore use and a fresh aliquot must be used for each coupling cycle. Theinitial color of all spots on the membrane was blue which is produced bybromophenol blue in the presence of the free amino groups on thede-protected amino acids.

[0049] As coupling proceeds with the addition of Fmoc-amino acid activeesters, the spots change to different colors for different amino acids.For example, Asparagine and Threonine change to green, Serine changes toyellow. The color change can be regarded as a sign that the coupling istaking place. After coupling an amino acid the membrane was washed 3×20mL DMF for 2 minutes each time to remove excess active esters.

[0050] Then, acetic anhydride was added to acetylate any uncoupled aminogroups to ensure no formation of deletion sequences. As all free aminogroups are capped by acetylation, the remaining blue color disappeared.The membrane was washed 3×20 mL DMF and then 20 mL of 20% piperidine inDMF was added to remove Fmoc protecting groups. After washing membrane5×20 mL DMF, 200 μL of 1% bromophenol blue solution was added to 20 mLDMF and this solution was added on the membrane. Due to piperidineremoval of the Fmoc groups, the spots turned blue leaving thesurrounding membrane white and the solution yellow. The membrane waswashed 3×20 mL with methanol. After air drying on a sheet of foldedfilter paper, the membrane is ready for the next coupling cycle. Thisprocedure was repeated for all but the final coupling cycle of thesynthesis.

[0051] For the final cycle, piperidine treatment was carried out rightafter the double coupling of active esters and DMF washing. Bromophenolblue solution was then added to obtain blue color for all spots andfinally the peptides on each spot were capped by acetylation. Aftersynthesis and acetylation, the protecting groups present on the sidechains of the amino acids must be relmoved. For side chain deprotection,5 mL of DCM was mixed with 5 mL TFA. The mixed solution was addedimmediately onto the air-dried membrane and the cleavage reaction wasallowed to proceed for 1 hour. The membrane was then washed with 3×20 mLDCM, 3×20 mL DMF, and 3×20 mL methanol. The membrane was air-dried andstored in a sealed plastic bag in the freezer (−20° C.) until requiredfor SPOTS analysis.

[0052] For analysis, the SPOTS membrane was first blocked with 20 mL ofTBS-blocking buffer overnight at room temperature. The membrane waswashed with 20 ml; Tris buffered saline (TBS) containing 0.05% Tween-20(T-TBS). The serum sample (Lyme disease or control) was diluted in 20 mLTBS-blocking buffer to 1:100. This diluted test antibody solution wasadded to the membrane and rocked for 3-4 hours at room temperature. Themembrane was washed with 3×20 mL T-TBS for 10 minutes each wash. Then,100 μL of P-galactosidase conjugated anti-human (G+M+A) secondaryantibody was diluted with 20 mL of TBS-blocking buffer. This was addedto the membrane and rocked for 2 hours at room temperature.

[0053] During this time, the signal development solution was prepared asfollows: Dissolve 4.9 mg BCIG in 100 μL DMF and 100 mg potassiumferricyanide in 1 mL MilliQ water. Add BCIG solution and 100 μL ofpotassium fenicyanide solution into 10 mL of phosphate buffered saline(PBS) containing 10 μL of 1 M magnesium chloride solution. After theincubation of the secondary antibody solution, wash the membrane 2×20 mLT-TBS followed by 2×20 mL PBS, then add the prepared signal developmentsolution to the membrane and rock at room temperature until blue spotsappear. Allow the color to develop for 40 to 50 minutes until a point atwhich there is a clear distinction between positive and negative spots.Pour off the signal development solution and wash the membrane with 2×20mL PBS. Photograph the stained membrane to provide a permanent record.

[0054] The SPOTS membrane must be regenerated after analysis of eachserum sample to remove I bound proteins before storage or re-probing. Toregenerate the membrane, it was washed with 5×20 mL MilliQ water andthen 3×20 mL DMF followed by another 2×20 mL MilliQ water. Then, 20 mL,of regeneration buffer A (485.0 g urea, 10.0 g SDS and 1 mL2-mercaptoetbanol in 1 L of MilliQ water) was added and the membrane wasincubated for 10 minutes at room temperature. The process was repeatedtwice. Then 20 mL of regeneration buffer B (Mix 400 mL of MilliQ waterand 500 mL ethanol, add 100 mL of acetic acid to above solution) wasadded and the membrane was incubated for 10 minutes at room temperature.The process was repeated twice. Finally, the membrane was washed with2×20 mL methanol and air-dried. The membrane was stored in a sealedplastic bag in the freezer (−20° C.) until the next analysis.

[0055] Svnthesis, Purification and Characterization of Epitope Peptides

[0056] All 7 epitope peptides (Table 1) were synthesized manually onPAL™ resin (0.34 mmol/g, 0.1-0.2 mm01 scale) in a polypropylene column(Bio-Rad Laboratories, Herculus, Calif.). DMF (3 ml) was added to swellthe resin for 20 min. After Fmoc de-protection with 20% piperidine inDMF for 2×20 min, the resin was rinsed with 3 ml of DMF three times, 3ml of methanol three times, then dried in air. The coupling was achievedby adding 3-fold molar excess of each amino acid, mixed with equimolaramounts of BOP and HOBt in 3 ml of DMF containing 1% (v/v) DHEA.Coupling proceeded at room temperature for 4 hours.

[0057] After coupling, the resin was washed with DMF and methanol andair-dried. A sample of the resin was tested with Kaiser ninhydrinreagent (1:1:1 v/v/v 0.2 mM KCN in pyridine, 4 mg/ml of phenol and 5%ninhydrin in butanol) at 10° C. for 3 min (Kaiser et al., 1970; Sarin etal., 1981). If the resin showed blue color, double coupling would beconducted for another 4 hours to drive the reaction to completion. Theresin was capped using 4 mL of DMF, 400 μL of acetic anhydride and 80 μLof triethylamine for 4 hours to eliminate any un-reacted amino groups.

[0058] The coupling procedure was repeated until the desired peptidesequence was obtained. When the assembly of the peptide sequence wascomplete, the N-terminus of all epitope peptides was capped with longchain biotin to serve two purposes simultaneously. The first purpose isto remove the charge associated with the free amino group of theN-terminus, thus to mimic the real environment in the natural proteinsequence. The second purpose is to use the biotin as the detection labelfor biotin-avidin binding in ELISA. TABLE 1 Synthesized Epitopes PeptideSequence FLA, VQEGVQQEGAQQP-(beta-A)(beta-,4)C 1639.8 AA 211-223 OspC2,EIAAKAIGKKIHQNNG-(beta-A)(beta-A)C 2274.3 AA 71-86 OspC3,ISTLIKQKLDGLKNE-(beta-A)(beta-A)C 2282.3 AA 104-118 OspC1O,PWAESPKKPE-(beta-A)(beta-A)C 1762.7 AA 198-207 P83-1,DKKAINLDKAQQKLD-(beta-A)(beta-A)C 2310.3 AA 296-310 P83-3,ITKGKSQKSLGD-(beta-A)(beta-A)C 1843.8 AA 431-442 P39,GMTFRAQEGAFLTG-(beta-A)(beta-A)C 2067.9 AA 129-142

[0059] Long chain biotin was selected to reduce any possibleconformational hindrance for high-avidity biotin-avidin binding. Allpeptides were cleaved from the resin with trifluoroacetic acid(TFA)/thioanisole/ethanedithiol (EDT)/anisole (90/5/3/2%, v/v) at 1mL/100 mg resin for 2 hours at room temperature. The cleavage mixturewas filtered through glass wool, which was then rinsed with TFA twice.The filtrates were combined and evaporated under an Argon stream toreduce the volume to about 1-2 mL, then precipitated by adding drop-wiseinto 10 times volume of ice-cooled diethyl ether. The white precipitatewas washed with cold diethyl ether five times to remove scavengers.Crude peptides were purified by reverse phase HPLC under acidiccondition (0.1% TFA), because cysteine was incorporated in all epitopepeptides for conjugation purpose and the availability of free thiolgroups in cysteine is critical for conjugating epitope peptides onto PLCcopolymer backbone. The acid condition can help to prevent or minimizethe oxidation of the free thiol groups. After HPLC purification, thetubes containing the epitope peptides were flushed with Argon stream,capped, wrapped with paraffin, and stored dry in the refrigerator (4°C.). The purified epitope peptides were characterized by amino acidanalysis and mass spectrometry.

[0060] Synthesis and Purification of Polyethylene Glycol-Aspartic AcidCopolymers

[0061] Amino group protected L-Aspartic acid (Boc-Asp-OH) (BACHEM, Kingof Prussia, Pa.) and α,ω-diamino-PEG (NH2-PEG-NH2, Shearwater Polymers,Huntsville, Ala.) were copolymerized based on carbodiimide reaction inthe presence of 4-(dimethyl amino)-pyridine (DMAP) and p-toluenesulfonicacid monohydrate (PTSA) as catalysts. In a typical preparation,NH2-PEG-NH2 (680 mg, 2×10 mol) and Boc-Asp-OH (46.6 mg, 2×10⁻⁴ mol) weredissolved in 20 mL methylene chloride with stirring. DMAP (12.2 mg,1×10⁻⁴ mol) and PTSA (19.0 mg, 1×10⁻⁴ mol) were added. To this solution1,3-diisopropylcarbodiimide (DIPC) (15.6 mL, 1×10⁻³ mol) was added at 0°C. under stirring. The reaction flask was sealed with a rubber stopperassembled with an Argon balloon. The reaction was allowed to continue atroom temperature with stirring until the reaction mixture becameviscous.

[0062] The reaction mixture was precipitated in 10 volumes of ice-coldethyl ether to obtain the white polymer product. The polymer was washedthree times with ice-cold ethyl ether and the polymer product wascollected by filtration or centrifugation. The polymer was dried underan Argon flow, re-dissolved in MilliQ water and purified by dialysisusing Spectra/For™ Spectrum cellulose ester membrane (MW 12-14,000 Da)for 24 h. After lyophilization, the polymer was treated with TFA for 3hours to remove all the Boc protecting groups. The de-protected polymersolution was then precipitated in 10 volumes of ice-cold ethyl ether,washed three times with ice-cold ethyl ether and dried under vacuum. Themolecular weight of the resulting PEG copolymer was measured by sizeexclusion chromatography.

[0063] Preparation of Polyethylene Glycol-Peptide Conjugates

[0064] To a solution of PEG copolymer in 50 mM carbonate-bicarbonatebuffer (pH=8.5) was added 0.5 equivalent (relative to the amino groupsin the polymer) of NHS-LC-Biotin in DMSO. The mixture was stirred atroom temperature under Argon overnight. After about 10 hours ofreaction, approximately 30% of the amino groups in the PEG, copolymerwere reacted and linked to biotin molecules. A fluorometric assay, usinga fluorogenic reagent, Fluram, was employed to check the extent of thebiotinylation reaction. In brief, 100 μL of PEG copolymer solution wassaved before adding the biotinylation reagent and diluted 10× in 0.2 Mborate buffer, pH 8.5) as reference. When reaction was complete, 100 mLof reaction mixture was taken and diluted 10× in 0.2 M borate buffer (pH8.5) as sample.

[0065] For fluorometric assay, 50 mL of Fluram solution (15 mg Fluramdissolved in 25 mL acetonitrile) was added to 150 μL of dilutedreference, 150 μL of diluted sample and 150 μL of blank (0.2 M boratebuffer, pH 8.5), respectively, in separate wells of a microtiter plate.After mixing immediately by pipetting up and down several times,fluorescence was read on a Fluorescence Multi-Well Plate Reader(CytoFluor™ 11, PerSeptive Biosystems) with the excitation wavelengthset at 400 nm and the emission wavelength set at 460 nm. The biotinlabeled PEG copolymer was purified by a Pharmacia Superdex-75 column andthen reacted with 3 molar equivalents of hetero-bifunctional NHS-PEG-VS(MW 2000 Da), relative to free amino groups remaining in biotin-labeledPEG copolymer.

[0066] The latter reaction, which was also monitored by the fluorometricassay, was complete after 4 hrs at room temperature (25° C.). Thefluorometric assay procedure was similar to that described above. Thefinal fluorescence reading was equal or close to the blank reading,suggesting that (all amino groups in the PEG copolymer had beensuccessfully derivatized. The reaction product was purified through aPharmacia Superdex-75 column or by membrane dialysis. For peptideconjugation, 5 molar equivalents of peptide relative to the availablevinylsulfone (VS) groups in the PEG copolymer were added to theactivated polymer solution, and these were allowed to react at 4° C.overnight. The final Biotin-PEG-peptide conjugate was purified by thePharmacia Superdex-75 column or by membrane dialysis, and concentratedto about 1 mg/mL using a Centricon™ ultrafilter (mw 10,000 Da). Aliquotswere stored as the stock antigen solution in the freezer (−20° C.) untilneeded.

[0067] The Enzyme-Linked Immuno-Sorbent Assay

[0068] ELISA is a simple but very sensitive immunoassay. It involves thefollowing basic steps: An antigen is bound to a solid phase material,usually a 96-well plastic plate. The solution containing the antibody tobe detected (usually serum) is added to the well having the immobilizedantigens. Unrelated, unbound antibody is then washed away. A secondantibody, which is an anti-immunoglobulin antibody linked with anenzyme, is then added to the wells. Then the substrate for the enzyme isadded to the above reaction mixture and the amount of enzymaticallyaltered substrate is measured. The enzyme and substrate are chosen sothat enzymatic modification of the substrate produces a change in colorof the substrate solution. The amount of changed substrate (which may bemeasured with a spectrophotometer) is proportional to the amount ofantibody bound to the immobilized antigen.

[0069] There are generally two types of ELISA formats: direct andindirect. In a direct ELISA, antigens first bind to the well surface ofthe plates, and then the bound antigens interact with the testantibodies and give the signals. In an indirect ELISA, the plates arefirst coated with antibodies that can capture antigens. The capturedantigens can then interact with the test antibodies and give thesignals.

[0070] Many modifications of the above basic technique can be useddepending on the nature of the sample, availability of reagents and theprecision and sensitivity required. For example, one may use abiotinylated antibody followed by enzyme-conjugated avidin orstreptavidin. The avidin-biotin method results in an amplified effectsince many biotin molecules may be attached to a single second antibodymolecule and multiple avidin molecules can then bind subsequently to thesecond antibody. For this reason, the avidin-biotin method isparticularly sensitive.

[0071] i) IgM Capture ELISA

[0072] In an IgM-capture format, IgM antibodies are captured or bound tothe test support, such as an ELISA plate. A representative portion ofall IgM antibodies, including disease specific and unrelated IgMantibodies, are captured. All other classes of antibodies are removed.

[0073] In a direct-capture test, the antigens are immobilized on thesurface of the plate. In an indirect-capture test, the antigens arepresent in the test solution and interact with the antibodies capturedor bound to the ELISA plate.

[0074] When the captured IgM antibodies are exposed to the preparedPEG-peptide conjugates, these Lyme disease specific epitope conjugateswill only bind to Lyme disease specific IgM antibodies. If no Lymedisease specific IgM antibodies are present, all conjugates will bewashed away and no signal can be detected. As a result, a negativeresult is obtained. Clearly, this indirect IgM capture ELISA format,combined with using the Lyme disease specific conjugates as antigens,increases the sensitivity and the specificity of detecting Lyme diseasespecific IgM antibodies, on which a highly sensitive and specificimmunoassay can be developed (FIG. 4).

[0075] ELISA plates were coated with 100 μL/well of affinity-purifiedgoat anti-human IgM antibody (10 μg/mL) in 0.04 M carbonate-bicarbonatebuffer, pH 9.6. Plates were slowly rotated on a Titer Plate Shaker(Lab-Line, Melrose Park, Ill.) for 2 h at room temperature, and kept at4° C. overnight. The plates were washed three times in a plate washer(ELP 3.5, Biotek, Winooski, Vt.) with PBS-B (10 mM phosphate bufferedsaline, 0.15 M sodium chloride, containing 0.1% BSA), blocked with 300μL/well of PBS-B milk (PBS-B containing 5% nonfat dry milk) for 2 h at37° C. Serum samples were diluted 1:100 in PBS-B milk, added at 100μL/well and rotated at 300 rpm for 1 h. The plates were washed fourtimes with PBS-B and incubated for 1 h with 100 1 μL/well ofBiotin-PEG-peptide conjugates (diluted to various concentrations inPBS-B milk).

[0076] During this time, the avidin-biotinylated peroxidase complex(ABC) was formed by adding one drop (50 μL) of reagent A (avidin DH) andone drop (50 μL) of reagent B (biotinylated peroxidase) to 5 mL ofPBS-BT (PBS-B containing 0.5 M sodium chloride and 0.1% Tween 20). TheABC reagent was vortexed and kept at room temperature for at least 30minutes before use. After washing the plates four times with PBS-B, 7 mLof PBS-BT was added to the ABC reagent and 100 μL of the diluted ABCreagent was-added to each well. The plate was rotated at 300 rpm for 30minutes and washed four times with PBS-B on the Biotek plate washerfollowed by two more manual washes with plain PBS. During the last wash,the two component 3,3′,5,5′-tetramethylbenzidine substrate solution(TMB) was prepared at room temperature. Substrate was added at 100μL/well with a repeater pipette (Eppendorf Plus/8), the plate wasrotated for 10 minutes to develop the color, and the reaction wasstopped by adding 100 μL/well of 1 M phosphoric acid. The plate was thenrotated for 2 more minutes to homogenize the color and then read on anELISA plate reader (Biotek) set for dual wavelengths (450 and 630 nm).

[0077] All seven Biotin-PEG-peptide conjugates were tested as antigensin IgM-capture ELISA individually and as in combination with a panel ofsamples containing sera from both Lyme disease patients and healthysubjects. A group of 12 negative control sera. were tested under thesame assay conditions and the average absorbance plus three standarddeviations of these control serum samples was used as the cutoff.

[0078] The index number of each serum sample was calculated as:Index=Absorbance of individual serum/Cutoff. An index number of 1.0 orabove is taken as a. positive and an index number of 0.8 or below istaken as a negative. Any index number between 0.8 to 1.0 is taken asequivocal.

[0079] ii) Clinical Diagnosis by IgM Capture ELISA

[0080] A panel of sera is tested by IgM capture ELISA using eitherprotein-based antigen (Borrelia burgdorferi sonicate) or ourpeptide-based antigens. The clinical diagnosis results are listed inTable 2.

[0081] The peptide-based ELISA using the combination of sevenPEG-peptide conjugates identified 31 positive samples from 33culture-proven positive samples, resulting in a diagnostic sensitivityof 94% (percentage of disease samples correctly diagnosed). Theprotein-based ELISA using sonicated Borrelia burgdorferi spirochetepicked up 23 samples out of 31 tested positive sera, yielding adiagnostic sensitivity of 74%. Furthermore, the peptide-based ELISA didnot yield any false positive results with the non-Lyme disease samplesgiving an essentially 100% of diagnostic specificity, whereas theprotein-based ELISA gave 6 false positives out of 23 negative samples,or a diagnostic specificity of 74% (percentage of non-disease samplescorrectly diagnosed). Thus, the peptide-based ELISA achieved highersensitivity and specificity than the protein-based ELISA.

[0082] As our design rationale predicted, the defined epitope peptidesshould have less tendency than whole proteins to cross-react with serafrom patients with other diseases, such as syphilis. In order to examinethis hypothesis further, a panel of serum samples from patients withsyphilis infection was tested using the combination of PEG-peptideconjugates (Table 3). Indeed, while 13 out of 25 syphilis samples gavecross-reactive results in the protein-ELISA, none of these testedsyphilis samples showed cross-reactivity in our peptide-ELISA whencorrected by subtracting serum background (no antigen used in ELISA),indicating that all seven epitope peptides defined in this study areLyme disease specific and do not cross-react with antibodies against thesyphilis spirochete.

SUMMARY

[0083] In our claims, we use the singular to include the plural (i.e.,“a” or “an” means “one or more”).

[0084] The present invention is not to be limited in scope by thespecific embodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodimentswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art and are intended to fall within the scope of the invention.Thus, for example, serum antibodies specific for any disease can beanalyzed in order to select disease-specific epitope sequences. Peptidescorresponding to these epitope sequences are then synthesized andconjugated in several copies to a multivalent PEG carrier molecule,along with a reporter group such as biotin. We thus intend the legalcoverage of our patent to be defined not by the scientific examples weinclude here, but by the legal claims appended here. TABLE 2 Comparisonof Lyme disease diagnosis for a panel of serum samples. Protein PeptideClinical No. ELISA ELISA diagnosis MC-2 P P P MC-3(6/18) N P PMC-3(6/26) P P P MC-4 P P P MC-7 P P P MC-8 N P P MC-9 P E P MC-10 P P PMC-14 P P P MC-17 P P P MC-23 P P P MC-33 P P P MC-41 P P P MC-59 P P PMC-62 P P P MC-68 N E P MC-70 P P P MC-71 B P P MC-72 P P P MC-73 P P PMC-74 N P P MC-91 N P P MC-92 P P P MC-93 N P P MC-100 N P P MC-101 P PP MC-JS P P P MC-GR P P P NC-1 P N N NC-2 N N N NC-3 N N N NC-4 N N NNC-5 N N N NC-8 N N N NC-9 N N N NC-10 N N N NC-11 N N N NC-14 N N NNC-15 N N N NC-16 N N N NC-A N N N NC-B N N N NC-C P N N NC-D N N N NC-BP N N NC-F N N N NC-G N N N NC-H N N N NC-LT P N N NC-LN P N N NC-LP P NN MC-SC P P P MC-EL P P P MC-AN P P P MC-MT ND P P MC-HA ND P P

[0085] TABLE 3 Comparison of ELISA results for serum samples frompatients with syphilis. Clinical No. Protein ELISA Peptide ELISAdiagnosis S-1 CR NCR Syphilis S-2 CR NCR Syphilis S-3 CR NCR SyphilisS-4 CR NCR Syphilis S-5 CR NCR Syphilis S-6 CR NCR Syphilis S-7 CR NCRSyphilis S-8 NCR NCR Syphilis S-9 CR NCR Syphilis S-10 NCR NCR SyphilisS-11 NCR NCR Syphilis S-12 NCR NCR Syphilis S-13 CR NCR Syphilis S-14NCR NCR Syphilis S-15 NCR NCR Syphilis S-16 NCR NCR Syphilis S-17 NCRNCR Syphilis S-18 NCR NCR Syphilis S-19 CR NCR Syphilis S-20 CR NCRSyphilis S-21 CR NCR Syphilis S-22 CR NCR Syphilis S-23 NCR NCR SyphilisS-24 NCR NCR Syphilis S-25 NCR NCR Syphilis

We claim:
 1. An immunological test kit including a composition of mattercomprising an immunologically reactive substance connected to animmunologically invisible carrier.
 2. The test kit of claim 1, whereinsaid immunological test kit is an ELISA kit.
 3. The test kit of claim 1,wherein said immunological test kit is an immuno-capillary kit.
 4. Thetest kit of claim 1, wherein said immunologically invisible carriercomprises polyethylene glycol.
 5. The test kit of claim 4, wherein saidimmunologically reactive substance consists essentially of an epitope.6. The test kit of claim 4, wherein said immunologically reactivesubstance comprises an antibody.
 7. The test kit of claim 4, whereinsaid immunologically reactive substance comprises an antigen.
 8. Thetest kit of claim 4, said composition of matter further comprising anattached reporter moiety.